ADP-ribosylation factor domain protein 1 (ARD1) initially cloned in the laboratory, is a member of the ADP-ribosylation factor (ARF) family of guanine nucleotide-binding proteins that differs from other ARFs by the presence of a 46-kDa amino-terminal extension (p5), which acts as a GTPase-activating protein (GAP) for its ARF domain (p3). Similar to ARF GAPs, the GAP domain of ARD1 contains a zinc finger motif and arginine residues that are critical for activity. It differs from other ARF GAPs in its covalent association with the GTP-binding domain and its specificity for the ARF domain of ARD1. ARFs are presumed to play a key role in the formation of intracellular transport vesicles and in their movement from one compartment to another. Both overexpressed and endogenous ARD1 were associated with Golgi and lysosomal membranes, consistent with a role in the formation or function of lysosomes and in protein trafficking between Golgi and lysosomes. The group had defined effects of numerous mutations in separately synthesized p3 and p5 on GTPase activity and protein-protein interactions, and described this year sequence motifs in p5 and p3 that serve, respectively, as signals for lysosomal and Golgi localization in cells. Interaction of ARD1 and cytohesin-1 was found in a yeast two-hybrid screen. Cytohesin-2 failed to interact in this system and had much less GEP activity toward ARD1 than did cytohesin-1, although they were equally active with ARF1. Preferential physical interaction was also shown in vitro and residues responsible for specificity of the cytohesin-1/ARD1 interaction were identified.